Abstracts

Rationale: The circadian regulator, CLOCK, was downregulated in surgical samples from patients with focal epilepsy. We have previously demonstrated that the Emx-cre;Clock-/- mice had sleep-related seizures. CLOCK and other circadian proteins were long thought to be an exclusively neuronal protein. However, recent studies demonstrated that Bmal1, the molecular binding partner of CLOCK, is also expressed in astrocytes. Additionally, since Emx-cre-recombination also affects astrocyte, it is possible that Clock deletion in cortical astrocytes contributes towards seizure generation. We sought out first to characterise if CLOCK is expressed in astrocytes.Metabolism is well-characterised to be circadian regulated and astrocyte metabolism contributed to epileptogenesis in various models of epilepsy. We hypothesised that CLOCK may regulate astrocyte metabolism and its dysfunction contributes to this epilepsy model. Specifically, we are interested in the contribution of pyridoxine metabolism. Pyridoxine metabolism is implicated in a model of epilepsy with knockout of the circadian transcription factors, PARbZip, which are regulated by CLOCK. We therefore wanted to examine if pyridoxine metabolism contributes in the CLOCK model of epilepsy. Methods: Primary astrocyte cultures were conducted in wildtype and Clock-/- P0-2 pups. Astrocytes were cultured in T25 flasks for 10-14 days prior to harvesting. Astrocyte culture media consists of DMEM with 25mM glucose, 1mM pyruvate, GlutaMAX, and 10% FBS. To detect CLOCK expressions, cells were harvested to obtain whole-cell lysate and Western blot was performed.To measure mitochondrial respiration, astrocytes were harvested and re-seeded in the Seahorse XFe24 plates at 3.5x104 cells. A Mito Stress Test assay was conducted to measure oxygen consumption rate (OCR) as indicators of various parameters of mitochondrial respiration, namely basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity. This assay was conducted in DMEM with 5mM glucose and 2.5mM pyruvate. Finally, to examine the contribution of pyridoxine metabolism, Seahorse assay media was supplemented with 100µM of the active form, pyridoxal phosphate (PLP), and the precursor, pyridoxal (PL). Results: CLOCK was found to be robustly expressed in astrocytes. This was confirmed both using Western blot and immunofluorescence. Clock-/- astrocytes exhibited significantly different mitochondrial respiration profile as compared to wildtype. This included significantly increased proton leak (p=0.0016), maximal respiration (p<0.0001), and spare respiratory capacity (p<0.0001). Basal respiration and ATP production was not significantly different (p>0.05). Supplementation with PLP attenuates the difference in maximal respiration and spare respiratory capacity but increases the difference in proton leak between Clock-/- and wildtype. Pyridoxal supplementation, however, attenuates the difference in all three respiration profiles. Both pyridoxal and pyridoxal phosphate supplementation did not affect basal respiration and ATP production. Conclusions: We demonstrated that CLOCK is indeed also expressed in astrocytes. Clock deletion in astrocytes affect mitochondrial respiration, especially pertaining to uncoupling capacity. Supplementation with pyridoxal and pyridoxal phosphate was able to rescue the metabolic dysfunction in the Clock-/- astrocytes. Future studies will seek to determine if pyridoxine supplementation can rescue the sleep-related seizures in the CLOCK-model of epilepsy. Funding: No funding